This invention is directed to compounds with heterocyclic-hydroxyimino-fluorene nuclei that mediate and/or inhibit the activity of certain protein kinases, and to pharmaceutical compositions containing such compounds. The invention is also directed to the therapeutic or prophylactic use of such compounds and compositions, and to methods of treating cancer, as well as other disease states associated with unwanted angiogenesis and/or cellular proliferation, by administering effective amounts of such compounds.
Protein kinases are a family of enzymes that catalyze phosphorylation of the hydroxyl group of specific tyrosine, serine, or threonine residues in proteins. Typically, such phosphorylation dramatically alters the function of the protein, and thus protein kinases are pivotal in the regulation of a wide variety of cellular processes, including metabolism, cell proliferation, cell differentiation, and cell survival. Of the many different cellular functions in which the activity of protein kinases is known to be required, some processes represent attractive targets for therapeutic intervention for certain disease states. Two examples are cell-cycle control and angiogenesis, in which protein kinases play a pivotal role; these processes are essential for the growth of solid tumors as well as for other diseases.
Uncontrolled cell proliferation is the insignia of cancer. Cell proliferation in response to various stimuli is manifested by a de-regulation of the cell division cycle, the process by which cells multiply and divide. Tumor cells typically have damage to the genes that directly or indirectly regulate progression through the cell-division cycle.
Cyclin-dependent kinases (CDKs) are serine-threonine protein kinases that play critical roles in regulating the transitions between different phases of the cell-cycle, such as the progression from a quiescent stage in G1 (the gap between mitosis and the onset of DNA replication for a new round of cell division) to S (the period of active DNA synthesis), or the progression from G2 to M phase, in which active mitosis and cell-division occurs. See, e.g., the articles compiled in Science, 274, 1643-1677 (1996); and Ann. Rev. Cell Dev. Biol., 13, 261-291 (1997). CDK complexes are formed through association of a regulatory cyclin subunit (e.g., cyclin A, B1, B2, D1, D2, D3, and E) and a catalytic kinase subunit (e.g., cdc2 (CDK1), CDK2, CDK4, CDK5, and CDK6). As the name implies, the CDKs display an absolute dependence on the cyclin subunit in order to phosphorylate their target substrates, and different kinase/cyclin pairs function to regulate progression through specific phases of the cell-cycle.
The progression from G1 to S phase, accomplished by the action of CDK4/cyclin D and CDK2/cyclin E, is subject to a variety of growth regulatory mechanisms, both negative and positive. Growth stimuli, such as mitogens, cause increased synthesis of cyclin D1 and thus increased functional CDK4. By contrast, cell growth can be down regulated in response to DNA damage or negative growth stimuli, by the induction of endogenous inhibitory proteins. These naturally occurring protein inhibitors include p21WAF1/CIP1, p27KIP1, and the p16INK4 family, the latter of which inhibit CDK4 exclusively (see Harper, Cancer Surv., 29, 91-107 (1997). Aberrations in this control system, particularly those that affect the function of CDK4 and CDK2, have been implicated in the advancement of cells to the highly proliferative state characteristic of malignancies, particularly familial melanomas, esophageal carcinomas, and pancreatic cancers. See, e.g., Hall et al., Adv. Cancer Res., 68, 67-108 (1996); Kamb, Trends in Genetics, 11, 136-140 (1995); Kamb et al., Science, 264, 436-440 (1994).
Over-expression of cyclin D1 is linked to esophageal, breast, and squamous cell carcinomas (see, e.g., DelSal et al., Critical Rev. Oncogenesis, 71, 127-142 (1996)). Genes encoding the CDK4-specific inhibitors of the p16 family frequently have deletions and mutations in familial melanoma, gliomas, leukemias, sarcomas, and pancreatic, non-small cell lung, and head and neck carcinomas (see Nobori et al., Nature, 368, 753-756 (1994)). Amplification and/or overexpression of cyclin E has also been observed in a wide variety of solid tumors, and elevated cyclin E levels have been correlated with poor prognosis. In addition, the cellular levels of the CDK inhibitor p27, which acts as both a substrate and inhibitor of CDK2/cyclin E, are abnormally low in breast, colon, and prostate cancers, and the expression levels of p27 are inversely correlated with the stage of disease (see Loda et al., Nature Medicine, 3, 231-234 (1997)). Recently there is evidence that CDK4/cyclin D might sequester p27, as reviewed in Sherr et al., Genes Dev., 13, 1501-1512 (1999). The p21 proteins also appear to transmit the p53 tumor-suppression signal to the CDKs (see El-Deiry et al., Cell, 75, 817-825 (1993)); thus, the mutation of p53 in approximately 50% of all human cancers may indirectly result in deregulation of CDK activity.
The use of compounds as anti-proliferative therapeutic agents that inhibit protein kinase activity is the subject of several patents and publications. For example, WIPO International Publication No. WO 97/45397 discloses certain alkyloxyamino-substituted fluorenones that control protein kinase C activity (e.g., CDC2 kinase activity) in mammals. WIPO International Publication No. WO 99/21845 discloses 4-aminothiazoles as CDK inhibitors. Isothiazole derivatives useful as anticancer agents are disclosed in WIPO International Publication No. WO 99/62890. U.S. Pat. No. 5,621,082 to Xiong et al. discloses nucleic acid derivatives that encode inhibitors of CDK6. Peptides and peptidomimetic inhibitors, including substrate site antagonists, are described in European Patent Publication No. 0 666 270 A2, Bandara et al., Nature Biotechnology, 15, 896-901 (1997), and Chen et al., Proc. Natl. Acad. Sci., USA, 96, 4325-4329 (1999). Peptide aptamers are identified in Cohen et al., Proc. Natl. Acad. Sci., U.S A., 95, 14272-14277 (1998). Other small molecules have been identified as CDK inhibitors (for recent reviews, see Webster, Exp. Opin. Invest. Drugs, 7, 865-887 (1998), Stover et al., Current Opinion in Drug Discovery and Development, 2, 274-285 (1999), and Rosania et al., Exp. Opin. Ther. Patents, 10, 215-230 (2000)). The flavone flavopiridol, displays modest selectivity for inhibition of CDKs over other kinases, but inhibits CDK4, CDK2, and CDK1 equipotently, with IC50s in the 0.1-0.3 xcexcM range. Flavopiridol is currently in Phase II clinical trials as an oncology chemotherapeutic (Stadler et al., J. Clin. Oncol., 18, 371-375 (2000) and Sedlacek et al., Int. J. Oncol., 9, 1143-1168 (1996)). Analogs of flavopiridol are the subject of other publications, for example, U.S. Pat. No. 5,733,920 to Mansuri et al. (WIPO International Publication No. WO 97/16447) and WIPO International Publication Nos. WO 97/42949, and WO 98/17662. Results of inhibition of CDKs with purine-based derivatives are described in Schow et al., Bioorg. Med. Chem. Lett., 7, 2697-2702 (1997); Grant et al., Proc. Amer. Assoc. Cancer Res,. 39, Abst. 1207 (1998); Legraverend et al., Bioorg. Med. Chem. Lett., 8, 793-798 (1998); Legraverend et al., J. Med. Chem., 43, 1282-1292 (2000); Gray et al., Science, 281, 533-538 (1998); Chang et al., Chemistry and Biology, 6, 361-375 (1999); and WIPO International Publication Nos. WO 99/02162, WO 99/43675, and WO 99/43676.
In addition, the following publications disclose certain pyrimidines that inhibit cyclin dependent kinases and growth-factor mediated kinases: WIPO International Publication Nos. WO 00/12485, WO 00/12486, and WO 98/33798; Ruetz et al., Proc. Amer. Assoc. Cancer Res., 39, Abst. 3796 (1998); and Meyer et al., Proc. Amer. Assoc. Cancer Res., 39, Abst. 3794 (1998). Benzensulfonamides that block cells in G1 are in development by Eisai, see Owa et al., J. Med. Chem., 42, 3789-3799 (1999). An oxindole CDK inhibitor is in development by Glaxo-Wellcome, see Luzzio et al., Proc. Amer. Assoc. Cancer Res., Abst. 4102 (1999) and WIPO International Publication No. WO 99/15500. Paullones were found in collaboration with the NCl, Schultz et al., J. Med. Chem., 2909-2019 (1999) and Kunick et al., Bioorg. Med. Chem. Lett., 10, 567-569 (2000). Indenopyrazoles are described in WIPO International Publication Nos. WO 99/17769 and WO 99/54308. Pyrazolo-pyridines are reported in WIPO International Publication No. WO 99/30710. Also known are the fluorene derivatives shown below in Comparison Examples 1 and 2; see also Pan et al., Chem. and Ind., 240-241 (1969), who disclose Comparison Example 2(a) and other 9-oxofluorene oximes: 
There is still a need, however, for other small-molecule compounds that may be readily synthesized and are potent inhibitors of one or more CDKs or CDK/cyclin complexes. See Gray et al., Curr. Med. Chem., 6, 859-875 (1999) and Sielecki et al., J. Med. Chem., 43, 1-18 (2000). Because CDK4 may serve as a general activator of cell division in most cells, and because complexes of CDK4/cyclin D and CDK2/cyclin E govern the early G1 phase of the cell-cycle, there is a need for effective and specific inhibitors of CDK4 and/or CDK2 for treating one or more types of tumors. Also, the pivotal roles of cyclin E/CDK2 and cyclin B/CDK1 kinases in the G1/S phase and G2/M transitions, respectively offer additional targets for therapeutic intervention in suppressing deregulated cell-cycle progression in cancer.
Another protein kinase, CHK-1, plays an important role as a checkpoint in cell-cycle progression. Checkpoints are control systems that coordinate cell-cycle progression by influencing the formation, activation and subsequent inactivation of the cyclin-dependent kinases. Checkpoints prevent cell-cycle progression at inappropriate times, maintain the metabolic balance of cells while the cell is arrested, and in some instances can induce apoptosis (programmed cell death) when the requirements of the checkpoint have not been met. See, e.g., Chen et al., Cell, 100, 681-692 (2000); O""Connor, Cancer Surveys, 29, 151-182 (1997); Nurse, Cell, 91, 865-867 (1997); Hartwell et al., Science, 266, 1821-1828 (1994); and Hartwell et al., Science, 246, 629-634 (1989).
One series of checkpoints monitors the integrity of the genome and, upon sensing DNA damage, these xe2x80x9cDNA damage checkpointsxe2x80x9d block cell-cycle progression in G1 and G2 phases, and slow progression through S phase. O""Connor, Cancer Surveys, 29, 151-182 (1997); Hartwell et al., Science, 266, 1821-1828 (1994). This action enables DNA repair processes to complete their tasks before replication of the genome and subsequent separation of this genetic material into new daughter cells takes place. Importantly, the most commonly mutated gene in human cancer, the p53 tumor suppressor gene, produces a DNA damage checkpoint protein that blocks cell-cycle progression in G1 phase and/or induces apoptosis (programmed cell death) following DNA damage. Hartwell et al., Science, 266, 1821-1828 (1994). The p53 tumor suppressor has also been shown to strengthen the action of a DNA damage checkpoint in G2 phase of the cell-cycle. See, e.g., Bunz et al., Science, 28, 1497-1501 (1998); Winters et al., Oncogene, 17, 673-684 (1998); Thompson, Oncogene, 15, 3025-3035 (1997).
Given the pivotal nature of the p53 tumor suppressor pathway in human cancer, therapeutic interventions that exploit vulnerabilities in p53-defective cancer have been actively sought. One emerging vulnerability lies in the operation of the G2 checkpoint in p53 defective cancer cells. Cancer cells, because they lack G1 checkpoint control, are particularly vulnerable to abrogation of the last remaining barrier protecting them from the cancer killing effects of DNA-damaging agents: the G2 checkpoint. The G2 checkpoint is regulated by a control system that has been conserved from yeast to humans. Important in this conserved system is a kinase, CHK-1, which transduces signals from the DNA-damage sensory complex to inhibit activation of the cyclin B/Cdc2 kinase, which promotes mitotic entry. See, e.g., Peng et al., Science, 277, 1501-1505 (1997); Sanchez et al., Science, 277, 1497-1501 (1997). Inactivation of CHK-1 has been shown to both abrogate G2 arrest induced by DNA damage inflicted by either anticancer agents or endogenous DNA damage, as well as result in preferential killing of the resulting checkpoint defective cells. See, e.g., Suganuma et al., Cancer Res., 59, 5887-5891 (1999); Nurse, Cell, 91, 865-867 (1997); Weinert, Science, 277, 1450-1451 (1997); Walworth et al., Nature, 363, 368-371 (1993); and Al-Khodairy et al., Molec. Biol. Cell 5, 147-160 (1994).
Selective manipulation of checkpoint control in cancer cells could afford broad utilization in cancer chemotherapeutic and radiotherapy regimens and may, in addition, offer a common hallmark of human cancer xe2x80x9cgenomic instabilityxe2x80x9d to be exploited as the selective basis for the destruction of cancer cells. A number of factors place CHK-1 as a pivotal target in DNA-damage checkpoint control. The elucidation of inhibitors of this and functionally related kinases such as CDS1/CHK-2, a kinase recently discovered to cooperate with CHK-1 in regulating S phase progression (see Zeng et al., Nature, 395, 507-510 (1998); Matsuoka, Science, 282, 1893-1897 (1998); Carr, Science, 287, 1765-1766 (2000); and Hirao et al., Science, 287, 1824-1827 (2000)), could provide valuable new therapeutic entities for the treatment of cancer.
Tyrosine kinases can be of the receptor type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular). At least one of the non-receptor protein tyrosine kinases, namely, LCK, is believed to mediate the transduction in T-cells of a signal from the interaction of a cell-surface protein (Cd4) with a cross-linked anti-Cd4 antibody. A more detailed discussion of non-receptor tyrosine kinases is provided in Bolen, Oncogene, 8, 2025-2031 (1993), which is incorporated herein by reference.
In addition to their role in cell-cycle control, protein kinases also play a crucial role in angiogenesis, which is the mechanism by which new capillaries are formed from existing vessels. When required, the vascular system has the potential to generate new capillary networks in order to maintain the proper functioning of tissues and organs. In the adult, however, angiogenesis is fairly limited, occurring only in the process of wound healing and neovascularization of the endometrium during menstruation. See Merenmies et al., Cell Growth and Differentiation, 8, 3-10 (1997). On the other hand, unwanted angiogenesis is a hallmark of several diseases, such as retinopathies, psoriasis, rheumatoid arthritis, age-related macular degeneneration, and cancer (solid tumors). See Folkman, Nature Med., 1, 27-31 (1995). Protein kinases which have been shown to be involved in the angiogenic process include three members of the growth factor receptor tyrosine kinase family: VEGF-R2 (vascular endothelial growth factor receptor 2, also known as KDR (kinase insert domain receptor) and as FLK-1); FGF-R (fibroblast growth factor receptor); and TEK (also known as Tie-2).
VEGF-R2, which is expressed only on endothelial cells, binds the potent angiogenic growth factor VEGF and mediates the subsequent signal transduction through activation of its intracellular kinase activity. Thus, it is expected that direct inhibition of the kinase activity of VEGF-R2 will result in the reduction of angiogenesis even in the presence of exogenous VEGF (see Strawn et al., Cancer Research, 56, 3540-3545 (1996)), as has been shown with mutants of VEGF-R2 which fail to mediate signal transduction. Millauer et al., Cancer Research, 56, 1615-1620 (1996). Furthermore, VEGF-R2 appears to have no function in the adult beyond that of mediating the angiogenic activity of VEGF. Therefore, a selective inhibitor of the kinase activity of VEGF-R2 would be expected to exhibit little toxicity.
Similarly, FGF-R binds the angiogenic growth factors aFGF and bFGF and mediates subsequent intracellular signal transduction. Recently, it has been suggested that growth factors such as bFGF may play a critical role in inducing angiogenesis in solid tumors that have reached a certain size. See Yoshiji et al., Cancer Research, 57, 3924-3928 (1997). Unlike VEGF-R2, however, FGF-R is expressed in a number of different cell types throughout the body and may or may not play important roles in other normal physiological processes in the adult. Nonetheless, systemic administration of a small molecule inhibitor of the kinase activity of FGF-R has been reported to block bFGF-induced angiogenesis in mice without apparent toxicity. See, e.g., Mohammadi et al., EMBO Journal, 17, 5896-5904 (1998).
TEK (also known as Tie-2) is another receptor tyrosine kinase expressed only on endothelial cells which has been shown to play a role in angiogenesis. The binding of the factor angiopoietin-1 results in autophosphorylation of the kinase domain of TEK and results in a signal transduction process which appears to mediate the interaction of endothelial cells with peri-endothelial support cells, thereby facilitating the maturation of newly formed blood vessels. The factor angiopoietin-2, on the other hand, appears to antagonize the action of angiopoietin-1 on TEK and disrupts angiogenesis. See Maisonpierre et al., Science, 277, 55-60 (1997).
As a result of the above-described developments, it has been proposed to treat angiogenesis by the use of compounds inhibiting the kinase activity of VEGF-R2, FGF-R, and/or TEK. For example, WIPO International Publication No. WO 97/34876 discloses certain cinnoline derivatives that are inhibitors of VEGF-R2, which may be used for the treatment of disease states associated with abnormal angiogenesis and/or increased vascular permeability such as cancer, diabetes, psoriosis, rheumatoid arthritis, Kaposi""s sarcoma, haemangioma, acute and chronic nephropathies, atheroma, arterial restinosis, autoimmune diseases, acute inflammation and ocular diseases with retinal vessel proliferation.
In addition to the protein kinases identified above, many other protein kinases have been considered to be therapeutic targets, and numerous publications disclose inhibitors of kinase activity, as reviewed in the following: McMahon et al., Oncologist, 5, 3-10 (2000); Garcia-Echeverria et al., Med. Res. Rev., 20, 28-57 (2000); Holash et al., Oncogene, 18, 5356-5362 (1999); Stover et al., Curr. Opin. Drug Disc. Dev., 2, 274-285 (1999); Toledo et al., Curr Med. Chem., 6, 775-805 (1999); Thomas et al., J. Biol. Chem., 274, 36684-36692 (1992); Cohen, Curr. Op. Chem. Biol., 10, 544-549 (1999); Adams et al., Curr. Opin. Drug Disc. Dev., 2, 96-109 (1999); McMahon et al., Curr. Opin. Drug Disc. Dev., 1, 131-146 (1998); and Strawn et al., Exp. Opin. Invest. Drugs, 7, 553-573 (1998).
There is still a need, however, for effective inhibitors of protein kinases. Moreover, as would be understood by those skilled in the art, it is desirable for kinase inhibitors to possess both high affinity for the target kinase as well as high selectivity versus other protein kinases.
Accordingly, an objective of the invention is to discover potent inhibitors of protein kinases. Another objective of the invention is to discover effective kinase inhibitors having a strong and selective affinity for a particular kinase.
These and other objectives of the invention, which will become apparent from the following description, have been achieved by the discovery of the heterocyclic-hydroxyimino-fluorene nuclei compounds, pharmaceutically acceptable prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts thereof (such compounds, prodrugs, metabolites and salts are collectively referred to as xe2x80x9cagentsxe2x80x9d) described below, which modulate and/or inhibit the activity of protein kinases. Pharmaceutical compositions containing such agents are useful in treating diseases mediated by kinase activity, such as cancer, as well as other disease states associated with unwanted angiogenesis and/or cellular proliferation, such as diabetic retinopathy, glaucoma, rheumatoid arthritis, restenosis, and psoriasis. Further, the agents have advantageous properties relating to the modulation and/or inhibition of the kinase activity associated with CDK complexes, CHK-1, CDS1, LCK, VEGF-R, and/or FGF-R.
In a general aspect, the invention relates to compounds of the Formula I: 
wherein:
R5 and R6 are each independently hydrogen, halo, or a substituted or unsubstituted C1-C8 alkyl, C1-C8 alkoxy, aryl, heteroaryl, acyl, thioalkyl, sulfonyl, or sulfoxyl; and
X is Cxe2x80x94Y or N, where Y is hydrogen, halo, NH2, NO2, or a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, alkoxy, alkenyl, aryl, heteroaryl, aryloxy, alkylamino, dialkylamino, thioalkyl, acyl, sulfonyl, sulfoxide, or thioaryl.
The invention is also directed to pharmaceutically acceptable prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts of the compounds of Formula I and their pharmaceutically active metabolites. Advantageous methods of making the compounds of the Formula I are also described.
In a preferred general embodiment, the invention relates to compounds having Formula I, wherein: R5 and R6 are each independently hydrogen, halo, or a substituted or unsubstituted C1-C8 alkyl; and X is Cxe2x80x94Y or N, where Y is hydrogen, halo, NH2, NO2, or a substituted or unsubstituted alkyl or aryl. In another preferred embodiment, the invention relates to compounds having Formula I, wherein: R5 and R6 are each independently hydrogen or halo; X is Cxe2x80x94Y or N, where Y is hydrogen, NH2, or NO2.
In another general aspect, the invention relates to compounds of the Formula II: 
wherein:
R5 and R6 are each independently hydrogen, halo, or a substituted or unsubstituted C1-C8 alkyl, C1-C8 alkoxy, aryl, heteroaryl, acyl, thioalkyl, sulfonyl, or sulfoxyl; and
W is O or S.
The invention is also directed to pharmaceutically acceptable prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts of the compounds of Formula II and their pharmaceutically active metabolites. Advantageous methods of making the compounds of the Formula II are also described.
In a preferred general embodiment, the invention relates to compounds having Formula II, wherein: R5 and R6 are each independently hydrogen, halo, or a substituted or unsubstituted C1-C8 alkyl; and W is O or S. In another preferred embodiment, the invention relates to compounds having Formula II, wherein: R5 and R6 are each independently hydrogen or halo; and W is O or S.
The invention also relates to a method of modulating and/or inhibiting the kinase activity of a CDK complex, VEGF-R, FGF-R, CHK-1, CDS1, and/or LCK by administering a compound of Formula I or II or a pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt of such compound or metabolite thereof. Preferably, compounds of the present invention have selective kinase activityxe2x80x94i.e., they possess significant activity against one specific kinase while possessing less or minimal activity against a different kinase.
The invention also relates to pharmaceutical compositions, each comprising an effective amount of an agent selected from compounds of Formula I and II and pharmaceutically active metabolites, pharmaceutically acceptable prodrugs, and pharmaceutically acceptable salts of such compounds and metabolites, and a pharmaceutically acceptable carrier or vehicle for such agent. The invention further provides methods of treating cancer as well as other disease states associated with unwanted angiogenesis and/or cellular proliferation, comprising administering effective amounts of one or more such agents to a patient in need of such treatment.
The inventive compounds of Formula I and II are useful for modulating the activity of protein kinases. More particularly, the compounds are useful as anti-angiogenesis agents and as agents for modulating and/or inhibiting the activity of protein kinases, thus providing treatments for cancer or other diseases associated with cellular proliferation mediated by protein kinases.
The terms xe2x80x9ccomprisingxe2x80x9d and xe2x80x9cincludingxe2x80x9d are used herein in their open, non-limiting sense.
The term xe2x80x9calkylxe2x80x9d as used herein refers to straight- and branched-chain alkyl groups having one to twelve carbon atoms. Exemplary alkyl groups include methyl (Me), ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like. The term xe2x80x9clower alkylxe2x80x9d designates an alkyl having from 1 to 8 carbon atoms (a C1-8-alkyl). Exemplary substituted alkyls include fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, and the like.
The term xe2x80x9calkenylxe2x80x9d refers to straight- and branched-chain alkenyl groups having from two to twelve carbon atoms. Illustrative alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, and the like.
The term xe2x80x9ccycloalkylxe2x80x9d refers to saturated carbocycles having from three to twelve carbon atoms, including bicyclic and tricyclic cycloalkyl structures. Exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
A xe2x80x9cheterocycloalkylxe2x80x9d group refers to a monocyclic radical containing carbon atoms, preferably 4 or 5 ring carbon atoms, and at least one heteroatom selected from nitrogen, oxygen and sulfur, and having no unsaturation.
The terms xe2x80x9carylxe2x80x9d (Ar) and xe2x80x9cheteroarylxe2x80x9d refer to monocyclic and polycyclic unsaturated or aromatic ring structures, with xe2x80x9carylxe2x80x9d referring to those that are carbocycles and xe2x80x9cheteroarylxe2x80x9d referring to those that are heterocycles. Examples of aromatic ring structures include phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, furyl, thienyl, pyrrolyl, pyridyl, pyridinyl, pyrazolyl, imidazolyl, pyrazinyl, pyridazinyl, 1,2,3-triazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1-H-tetrazol-5-yl, indolyl, quinolinyl, benzofuranyl, benzothiophenyl (thianaphthenyl), and the like. Such moieties may be optionally substituted by one or more suitable substituents, for example, a substituent selected from a halogen (F, Cl, Br or I); lower alkyl; OH; NO2; CN; CO2H; O-lower alkyl; aryl; aryl-lower alkyl; CO2CH3; CONH2; OCH2CONH2; NH2; SO2NH2; OCHF2; CF3; OCF3; and the like. Such moieties may also be optionally substituted by a fused-ring structure or bridge, for example OCH2xe2x80x94O.
The term xe2x80x9calkoxyxe2x80x9d refers to the radical xe2x80x94O-alkyl. Illustrative examples include methoxy, ethoxy, propoxy, and the like.
The term xe2x80x9caryloxyxe2x80x9d respresents xe2x80x94O-aryl, wherein aryl is defined above.
The term xe2x80x9chalogenxe2x80x9d represents chlorine, fluorine, bromine or iodine. The term xe2x80x9chaloxe2x80x9d represents chloro, fluoro, bromo or iodo.
The term xe2x80x9calkylaminoxe2x80x9d represents xe2x80x94NHR where R is an alkyl group as defined above.
The term xe2x80x9cdialkylaminoxe2x80x9d represents xe2x80x94NHRaRb where RaRb are each independently an alkyl group as defined above.
The term xe2x80x9cthioalkylxe2x80x9d refers to the radical xe2x80x94SR where R is an alkyl group as defined above.
The term xe2x80x9cacylxe2x80x9d represents xe2x80x94C(O)H, xe2x80x94C(O)OH, xe2x80x94C(O)R, xe2x80x94C(O)OR, xe2x80x94C(O)NH2, xe2x80x94C(O)NHR, and xe2x80x94C(O)NHRaRb, where R and RaRb are as defined above.
The term xe2x80x9cthioarylxe2x80x9d refers to the radical xe2x80x94SAr where Ar is an aryl group as defined above.
The term xe2x80x9csulfonylxe2x80x9d represents the radical xe2x80x94SO2R or xe2x80x94SO2Ar, where R is an alkyl group and Ar is an aryl group as defined above.
The term xe2x80x9csulfoxylxe2x80x9d represents the radical xe2x80x94SOR or xe2x80x94SOAr, where R is an alkyl group and Ar is an aryl group as defined above.
As indicated, the various moieties or functional groups for variables in the formulae may be optionally substituted by one or more suitable substituents. Exemplary substituents include a halogen (F, Cl, Br, or I), lower alkyl, xe2x80x94OH, xe2x80x94NO2, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94O-lower alkyl, -aryl, -aryl-lower alkyl, xe2x80x94CO2CH3, xe2x80x94CONH2, xe2x80x94OCH2CONH2, xe2x80x94NH2, xe2x80x94SO2NH2, haloalkyl (e.g., xe2x80x94CF3, xe2x80x94CH2CF3), xe2x80x94O-haloalkyl (e.g., xe2x80x94OCF3, xe2x80x94OCHF2), and the like.
The compounds of the invention may exhibit the phenomenon of tautomerism. While Formula I and II cannot expressly depict all possible tautomeric forms, it is to be understood that Formula I and II are intended to represent any tautomeric form of the depicted compound and are not to be limited merely to a specific compound form depicted by the formula drawings. For example, Formula I may tautomerize to the following structure: 
It is also understood that a compound of Formula I or II may exist as an xe2x80x9cExe2x80x9d or xe2x80x9cZxe2x80x9d configurational isomer, or a mixture of E and Z isomers. For example, an E isomer exists when the hydroxy (xe2x80x94OH) substituent of the oxime is on the opposite side of the heterocyclic-portion of a compound depicted in Formula I, wherein a Z isomer exists when the hydroxy (xe2x80x94OH) substituent is on the same side as the heterocyclic-portion of a compound, as expressly depicted in Formula I. A mixture of E and Z isomers is indicated by a wavy bond between the nitrogen atom and hydroxy substituent, as expressly depicted in Examples A-F. It is therefore to be understood that Formula I and II are intended to represent any configurational form of the depicted compound and is not to be limited merely to a specific compound form depicted by the formula drawings.
Some of the inventive compounds may exist as single stereoisomers (i.e., essentially free of other stereoisomers), racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the present invention. Preferably, the inventive compounds that are optically active are used in optically pure form.
As generally understood by those skilled in the art, an optically pure compound having one chiral center (i.e., one asymmetric carbon atom) is one that consists essentially of one of the two possible enantiomers (i.e., is enantiomerically pure), and an optically pure compound having more than one chiral center is one that is both diastereomerically pure and enantiomerically pure. Preferably, the compounds of the present invention are used in a form that is at least 90% optically pure, that is, a form that contains at least 90% of a single isomer (80% enantiomeric excess (xe2x80x9ce.e.xe2x80x9d) or diastereomeric excess (xe2x80x9cd.e.xe2x80x9d)), more preferably at least 95% (90% e.e. or d.e.), even more preferably at least 97.5% (95% e.e. or d.e.), and most preferably at least 99% (98% e.e. or d.e.).
Additionally, the formulas are intended to cover solvated as sell as unsolvated forms of the identified structures. For example, Formula I includes compounds of the indicated structure in both hydrated and non-hydrated forms. Other examples of solvates include the structures in combination with isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.
In addition to compounds of Formulas I and II, the invention includes pharmaceutically acceptable prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts of such compounds and metabolites.
xe2x80x9cA pharmaceutically acceptable prodrugxe2x80x9d is a compound that may be converted under physiological conditions or by solvolysis to the specified compound or to a pharmaceutically acceptable salt of such compound.
xe2x80x9cA pharmaceutically active metabolitexe2x80x9d is intended to mean a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof.
Prodrugs and active metabolites of a compound may be identified using routine techniques known in the art. See, e.g., Bertolini et al., J. Med. Chem., 40, 2011-2016 (1997); Shan, et al., J. Pharm. Sci., 86 (7), 765-767; Bagshawe, Drug Dev. Res., 34, 220-230 (1995); Bodor, Advances in Drug Res., 13, 224-331 (1984); Bundgaard, Design of Prodrugs (Elsevier Press 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991).
xe2x80x9cA pharmaceutically acceptable saltxe2x80x9d is intended to mean a salt that retains the biological effectiveness of the free acids and bases of the specified compound and that is not biologically or otherwise undesirable. A compound of the invention may possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. Exemplary pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base, such as salts including sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, xcex3-hydroxybutyrates, glycolates, tartrates, methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.
If the inventive compound is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.
If the inventive compound is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
In the case of agents that are solids, it is understood by those skilled in the art that the inventive compounds and salts may exist in different crystal or polymorphic forms, all of which are intended to be within the scope of the present invention and specified formulas.
Therapeutically effective amounts of the agents of the invention may be used to treat diseases mediated by modulation or regulation of protein kinases. An xe2x80x9ceffective amountxe2x80x9d is intended to mean that amount of an agent that, when administered to a mammal in need of such treatment, is sufficient to effect treatment for a disease mediated by the activity of one or more protein kinases, such as tryosine kinases. Thus, e.g., a therapeutically effective amount of a compound of the Formula I, salt, active metabolite or prodrug thereof is a quantity sufficient to modulate, regulate, or inhibit the activity of one or more protein kinases such that a disease condition which is mediated by that activity is reduced or alleviated.
The amount of a given agent that will correspond to such an amount will vary depending upon factors such as the particular compound, disease condition and its severity, the identity (e.g., weight) of the mammal in need of treatment, but can nevertheless be routinely determined by one skilled in the art. xe2x80x9cTreatingxe2x80x9d is intended to mean at least the mitigation of a disease condition in a mammal, such as a human, that is affected, at least in part, by the activity of one or more protein kinases, such as tyrosine kinases, and includes: preventing the disease condition from occurring in a mammal, particularly when the mammal is found to be predisposed to having the disease condition but has not yet been diagnosed as having it; modulating and/or inhibiting the disease condition; and/or alleviating the disease condition.
The affinity of the compounds of the invention for a receptor may be enhanced by providing multiple copies of the ligand in close proximity, preferably using a scaffolding provided by a carrier moiety. It has been shown that provision of such multiple valence compounds with optimal spacing between the moieties dramatically improves binding to a receptor. See, e.g., Lee et al., Biochemistry, 23, 4255 (1984). The multivalency and spacing can be controlled by selection of a suitable carrier moiety or linker units. Such moieties include molecular supports which contain a multiplicity of functional groups that can be reacted with functional groups associated with the compounds of the invention. Of course, a variety of carriers can be used, including proteins such as BSA (bovine serum albumin) or HSA (human serum albumin), various peptides including, for example, pentapeptides, decapeptides, and pentadecapeptides, and the like. The peptides or proteins can contain the desired number of amino acid residues having free amino groups in their side chains; however, other functional groups, such as sulfhydryl groups or hydroxyl groups, can also be used to obtain stable linkages.
Agents that potently regulate, modulate, or inhibit the protein kinase activity associated with receptors CDK complexes, VEGF, FGF, CHK-1, CDS1, and LCK, among others, and which inhibit angiogenesis and/or cellular profileration are preferred. The present invention is further directed to methods of modulating or inhibiting protein kinase activity, for example in mammalian tissue, by administering an inventive agent. The activity of the inventive agents as modulators of protein kinase activity, such as the activity of kinases, may be measured by any of the methods available to those skilled in the art, including in vivo and/or in vitro assays. Examples of suitable assays for activity measurements include those described in WIPO International Publication No. WO 99/21845; Parast et al., Biochemistry, 37, 16788-16801 (1998); Jeffrey et al., Nature, 376, 313-320 (1995); WIPO International Publication No. WO 97/34876; and WIPO International Publication No. WO 96/14843. These properties may be assessed, for example, by using one or more of the biological testing procedures set out in the examples below.
The active agents of the invention may be formulated into pharmaceutical compositions as described below. Pharmaceutical compositions of this invention comprise an effective modulating, regulating, or inhibiting amount of a compound of Formula I or Formula II and an inert, pharmaceutically acceptable carrier or diluent. In one embodiment of the pharmaceutical compositions, efficacious levels of the inventive agents are provided so as to provide therapeutic benefits involving modulation of protein kinases. By xe2x80x9cefficacious levelsxe2x80x9d is meant levels in which the effects of protein kinases are, at a minimum, regulated. These compositions are prepared in unit-dosage form appropriate for the mode of administration, e.g., parenteral or oral administration.
An inventive agent can be administered in conventional dosage form prepared by combining a therapeutically effective amount of an agent (e.g., a compound of Formula I) as an active ingredient with appropriate pharmaceutical carriers or diluents according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
The pharmaceutical carrier employed may be either a solid or liquid. Exemplary of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time-delay or time-release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
A variety of pharmaceutical forms can be employed. Thus, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation will be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension.
To obtain a stable water-soluble dose form, a pharmaceutically acceptable salt of an inventive agent is dissolved in an aqueous solution of an organic or inorganic acid, such as 0.3 M solution of succinic acid or citric acid. If a soluble salt form is not available, the agent may be dissolved in a suitable cosolvent or combinations of cosolvents. Examples of suitable cosolvents include, but are not limited to, alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, gylcerin and the like in concentrations ranging from 0-60% of the total volume. In an exemplary embodiment, a compound of Formula I is dissolved in DMSO and diluted with water. The composition may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution.
It will be appreciated that the actual dosages of the agents used in the compositions of this invention will vary according to the particular complex being used, the particular composition formulated, the mode of administration and the particular site, host and disease being treated. Optimal dosages for a given set of conditions can be ascertained by those skilled in the art using conventional dosage-determination tests in view of the experimental data for an agent. For oral administration, an exemplary daily dose generally employed is from about 0.001 to about 1000 mg/kg of body weight, with courses of treatment repeated at appropriate intervals. Administration of prodrugs are typically dosed at weight levels which are chemically equivalent to the weight levels of the fully active form.
The compositions of the invention may be manufactured in manners generally known for preparing pharmaceutical compositions, e.g., using conventional techniques such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, which may be selected from excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
Proper formulation is dependent upon the route of administration chosen. For injection, the agents of the invention may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks""s solution, Ringer""s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with the active ingredient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration intranasally or by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit-dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
For administration to the eye, a compound of Formula I or Formula II is delivered in a pharmaceutically acceptable ophthalmic vehicle such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye, including, for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/cilary, lens, choroid/retina and scelera. The pharmaceutically acceptable ophthalmic vehicle may be an ointment, vegetable oil, or an encapsulating material. A compound of the invention may also be injected directly into the vitreous and aqueous humor.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g, containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described above, the compounds may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
An examplary pharmaceutical carrier for hydrophobic compounds is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be a VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) contains VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid- or gel-phase carriers or excipients. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Some of the compounds of the invention may be provided as salts with pharmaceutically compatible counter ions. Pharmaceutically compatible salts may be formed with many acids, including hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free-base forms.
The inventive agents may be prepared using the reaction routes and synthesis schemes as described below, employing the general techniques known in the art using starting materials that are readily available. The preparation of preferred compounds of the present invention is described in detail in the following examples, but the artisan will recognize that the chemical reactions described may be readily adapted to prepare a number of other protein kinase inhibitors of the invention. For example, the synthesis of non-exemplified compounds according to the invention may be successfully performed by modifications apparent to those skilled in the art, e.g., by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions. Alternatively, other reactions disclosed herein or generally known in the art will be recognized as having applicability for preparing other compounds of the invention.